The kidney is one of the major target organs for drug-induced toxicity. Nephrotoxic drugs and chemicals can induce acute kidney injury (AKI), or chronic kidney disease and subsequently end stage renal disease (ESRD) (1-3). AKI and ESRD patients have increased morbidity and mortality and depend on dialysis (1, 4, 5). About 5% of all hospitalized patients and ˜20%-30% of ICU patients develop AKI, and ˜20%-25% of these cases are due to nephrotoxic drugs (2-4). When alternative and new drugs become available their nephrotoxic potential is often underestimated (6), which leads again to clinical complications, as in case of COX2 inhibitors (7).
Typically, nephrotoxicity is detected only late during drug development and accounts for 2% of drug attrition during pre-clinical studies and 19% in phase 3 (8). Also, due to the large functional reserve of the kidney nephrotoxic effects often become obvious only after regulatory approval. A recent example is tenofovir, which injures the renal proximal tubules (9, 10). Together, the problems outlined above are associated with increased risks for patients and subjects enrolled in clinical trials as well as substantial costs for the health care system and the pharmaceutical industry.
One major problem is the lack of pre-clinical models with high predictability. The predictability of animal models is compromised by interspecies variability, and there are other problems such as high costs and low throughput. Further, legislation changes in the EU (REACH and the 7th Amendment of the Cosmetics Directive) and new initiatives in the USA (ToxCast and Tox21) have increased the interest in in vitro models. Regulatory accepted or validated in vitro models for the prediction of nephrotoxicity in humans are currently not available. Major difficulties are related to the identification of appropriate cell types and endpoints (11-13).
In the kidney the cells of the renal proximal tubule (PT) are a major target for drug-induced toxicity due to their roles in glomerular filtrate concentration and the transport of drugs and organic compounds (2, 3). PT-derived cell lines, such as the human and porcine cell lines HK-2 (human kidney-2) and LLC-PK1 (Lewis lung cancer-porcine kidney 1), have been frequently applied in in vitro nephrotoxicology. However, immortalized cells are less sensitive than human primary renal proximal tubular cells (HPTC) (14) and insensitive to well-known nephrotoxicants (13), which is due to do functional changes and changes in drug transporter expression associated with immortalization (15-17). Further, endpoints that are associated with general cytotoxicity, such as cell death, metabolic activity or ATP depletion, are not useful for addressing organ-specific toxicity. A recent study measuring ATP-depletion in liver-, kidney PT- and heart-derived cell lines treated with hepatotoxic, nephrotoxic and cardiotoxic compounds found that the majority of compounds had similar effects in all three cell lines (18).
The European and US regulatory agencies in charge of the validation and acceptance of alternative methods (European Centre for the Validation of Alternative Methods (ECVAM) and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods/Interagency Coordinating Committee on the Validation of Alternative Methods (NICEATM/ICCVAM)) are currently not involved in any activities on the validation of methods for in vitro nephrotoxicology. The ECVAM has funded one pre-validation study (19) which used 15 drugs. Other models for in vitro nephrotoxicology that have been developed since then during the last 10 years (20-24) have been tested with limited numbers of drugs and are of unclear predictability. A recently developed high-throughput mitochondrial nephrotoxicant assay is based on rabbit cells (25), which raises issues concerning interspecies variability. This applies also to a model employing PT freshly isolated from murine kidneys (23, 24). Both models would still require the use of animals.